Detecting the products of enzymatic reactions, e.g., DNA polymerase reactions, ligase reactions, kinase reactions, phosphatase reactions, and others, is central to molecular and cell biology, genomic analysis, diagnostic medicine, pharmaceutical research, and many other fields of science and medicine. By linking a highly visible signal to a component in an enzymatic reaction, one can better monitor the production, consumption, and/or conversion of reactants and/or products. This strategy can also assist one in identifying any potential effectors or inhibitor of the reaction. Optical labels (e.g., labels having moieties with high quantum yields, such as fluorescent or luminescent moieties) predominate as analytical tools. The widespread adoption of optical labeling methodologies is attributable to their sensitivity and ease of detection, their relative handling safety, and the ease with which they can be integrated with available detection systems (e.g., using microscopes, cameras, photomultipliers, CCD arrays and combinations thereof). For example, high-throughput analysis systems in which optical labels are frequently used include DNA sequencers, array readout systems, cell analysis and sorting systems, and the like. For a brief overview of optical labels, fluorescent products, and technologies see, e.g., Sullivan (ed) (2007) Fluorescent Proteins, Volume 85, Second Edition (Methods in Cell Biology) ISBN-10: 0123725585; Hof et al. (eds) (2005) Fluorescence Spectroscopy in Biology: Advanced Methods and their Applications to Membranes, Proteins, DNA, and Cells (Springer Series on Fluorescence) ISBN-10: 354022338X; Haughland (2005) Handbook of Fluorescent Probes and Research Products, 10th Edition (Invitrogen, Inc./Molecular Probes); BioProbes Handbook, (2002) from Molecular Probes, Inc.; and Valeur (2001) Molecular Fluorescence: Principles and Applications Wiley ISBN-10: 352729919X.
The detection of optical labels in an enzymatic reaction generally entails directing an excitation radiation source at the reaction mixture to excite the labeling group present in the mixture, which is then separately detectable. However, prolonged exposure of chemical and biochemical reactants to radiation (e.g., light) energy during the excitation and detection of optical labels can damage e.g., enzymes, proteins, substrates, or the like, in the reaction mixture. For example, it has been observed that in template-directed synthesis of nucleic acids comprising fluorescently labeled nucleotides or nucleotide analogs, sustained exposure of the DNA polymerase to excitation radiation used in the detection of the relevant label (e.g., fluorophore) reduces the enzyme's processivity and polymerase activity. Typically, illuminated reactions proceed under conditions wherein the reactants (e.g., enzyme molecules, etc.) are present in excess, such that any adverse effects of photodamage on, e.g., any single enzyme molecule in the reaction mix, do not, in general, affect the operation of the assay.
An increasing number of analyses that entail the use of optical labels are performed with reactants at very low concentrations. For example, polymerases can be used to synthesize DNAs that comprise fluorescently labeled nucleotide analogs in microfluidic or nanofluidic reaction vessels or channels, or in single molecule analyses, e.g., in optically confined reaction volumes, e.g., in a zero-mode waveguide (ZMW) or ZMW array. Analysis of small, single-analyte reaction volumes is becoming increasingly important in high-throughput applications, e.g., in DNA sequencing. However, in such reactant-limited analyses, any degradation of a critical reagent, e.g., an enzyme molecule, due to photodamage, can dramatically interfere with the analysis, e.g., a single-molecule sequencing reaction, by further limiting the reagent.
Enzymes, e.g., DNA polymerases, that exhibit decreased sensitivity to photodamage are desirable for use in a variety of single- or low-number enzyme analyses, including, e.g., DNA sequencing, nucleic acid amplification, labeling reactions, analyte detection assays, kinase assays, phosphatase assays, and others. What are needed in the art are enzymes that exhibit improved tolerance to fluorescence-generated reactive species. What are also needed are methods of making and using such enzymes. The invention described herein fulfills these and other needs, as will be apparent upon review of the following.